One mechanism by which cells detect the presence of cytosolic viruses is through retinoic acid–inducible gene I (RIG-I), which binds to viral RNA and associates with the mitochondrial outer membrane adaptor protein MAVS. MAVS activates the transcription factors nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF-3), which induce production of the type I interferons required for the antiviral response. However, the mechanism by which MAVS activates IRF-3 is incompletely understood. Noting that ubiquitination is thought to regulate the RIG-I pathway, Zeng et al. devised a cell-free system in which mitochondria isolated from uninfected or infected HEK 293T cells were incubated with cytosolic extracts from uninfected cells. Phosphorylated and dimerized (and therefore activated) IRF-3 was detected in cytosolic extracts incubated with mitochondria from infected HEK 293T cells. Purification of factors responsible for IRF-3 activation from cytosolic fractions led to the identification of the E2 ubiquitin-conjugating enzyme Ubc5. Experiments in cells treated with specific short hairpin RNAs (shRNAs) showed that catalytically active Ubc5 was required for RIG-I- and MAVS-dependent activation of IRF-3. Tests of a panel of ubiquitin mutants showed that Lys63-mediated, but not Lys48-mediated, polyubiquitination was required for the activation of IRF-3; however, the E3 ubiquitin ligase involved was not identified. Consistent with a study that implicated inhibitor of NF-κB kinase γ (IKKγ, also known as NEMO) in the RIG-I to IRF-3 pathway, experiments in the cell-free system showed that two ubiquitin-binding domains of NEMO were required for the activation of IRF-3. Together, these data suggest that Lys63-mediated polyubiquitination is required to activate IRF-3 as part of the antiviral immune response.